Several methods have been described for the production of antihemophilic factor (AHF or Factor VIII) for therapeutic use. Such methods generally start from blood plasma cryoprecipitates and involve the extraction of Factor VIII by suitable extraction agents, possibly followed by purification steps.
One of said methods is described in British patent specification No. 1,551,928 and comprises the steps of extracting cryoprecipitate in pyrogen-free water at room temperature, removing denatured proteins and prothrombin complex from the extracted solution by adsorption, precipitating fibrinogen from the low ionic strength liquid extract by cooling the liquid to about 1.degree. C. to 2.degree. C., and lyophilizing the supernatant liquid to obtain the Factor VIII concentrate.
The above-mentioned method results in a liquid concentrate with a Factor VIII concentration generally comprised between 10 and 14 I.U. (International Units) per ml. However there is a need for higher concentrations for several reasons. For example, it would be very desirable to obtain 100 ml vials containing 1000 units of lyophilized Factor VIII but the volume of liquid Factor VIII concentrate to be dried would be too large to allow use of such vials.
Concentrating such product should be possible by dissolving a lyophilized powder in a small volume and then re-drying, or by the use of ultra-filtration. But the high proteic content of the resulting solutions would give a final lyophilized powder with poor solubility.
Another of such methods is described in the Wickerhauser U.S. Pat. No. 4,104,266 and provides a first extraction of a cryoprecipitate with a low ionic strength buffer solution comprising tris(hydroxymethyl)aminomethane at a temperature of about 0.degree. C. to obtain a cold insoluble fraction having cold soluble impurities removed therefrom, followed by the step of extracting said cold insoluble fraction with a similar buffer solution at a temperature of about 21.degree. C. to obtain a solution comprising the Factor VIII. In fact the Factor VIII concentration before lyophilizing seems limited to about 8 to 10 I.U. per ml.
It is an object of the present invention to provide a new and useful improvement for preparation of a Factor VIII concentrate from a cryoprecipitate whereby a higher Factor VIII concentration is obtained of at least 20 to 25 I.U. per ml.
Another object of the present invention is to obtain lyophilized Factor VIII vials containing at least 1000 to 1400 I.U. per 100 ml. vial.
Another object of the present invention is to obtain such lyophilized Factor VIII allowing a short dissolution time, even when reducing the amount of fluid used for reconstitution in order to shorten infusion time.
Another object of the present invention is to increase the removal of proteic impurities, including fibrinogen, without significantly decreasing the final yield of Factor VIII.
Another object of the present invention is to provide such an improvement able to be performed on an industrial scale for treating cryoprecipitate collected from more than 100 l and even more than 1000 l of plasma.
Another object of the present invention is to improve a method of preparation of a Factor VIII concentrate according to British patent specification No. 1,551,928.